Polynucleotide fragments of an infectious human endogenous retrovirus

ABSTRACT

Described is a polynucleotide molecule being a fragment of a polynucleotide sequence of an infectious human endogenous retrovirus, said polynucleotide molecule comprising a sequence selected from the group consisting of (a) a sequence with at least 98%, preferably 99%, still preferred 99.5%, identity to a sequence according to SEQ ID No 1, (b) a sequence which hybridises under stringent conditions with a nucleotide sequence according to said SEQ ID No 1, (c) a sequence which differs from said sequences (a) or (b) due to degeneration of the genetic code and (d) a sequence comprising sequences inserted into vectors pCR4-Topo and deposited as “MERV-env”, “MERV-gag”, “MERV-prt” and “MERV-pol” at the DSMZ on 26 Sep. 2001 or fragments thereof.

This application is a national phase application under 35 U.S.C. § 371of International Application No. PCT/EP02/10899 filed 27 Sep. 2002,which claims priority to Austrian Application No. A 1539/2001 filed 27Sep. 2001, the contents of which are incorporated herein by reference intheir entirety.

The present invention relates to human endogenous retroviruses,fragments thereof, a biologically functional vector comprising sequencesof said retrovirus.

Endogenous retroviruses (ERVs) are integral parts in the genomes ofmany, if not of all, species. ERVs most probably resulted from infectionof germ line cells with exogenous retroviruses and subsequent fixationof their genetic information in the host genome.

Many types of human endogenous retroviruses (HERVs) have beencharacterised previously, and they have been classified into differentgroups, or families, partly on the basis of their sequence identity andpartly according to the similarity of their primer binding sites (PBSs)to host tRNAs. Thus, members of the HERV.H family contain a PBS with asequence similar to a region of tRNA^(His), whereas the HERV.E family isprimed by tRNA^(Glu). Despite the large amount of data available, theclassification of the many different HERV families within an overallphylogenetic framework has been hampered for several reasons: (i) somehighly divergent retroviruses are primed by the same type of tRNA; (ii)many HERV families have not been fully characterised, and the sequenceinformation that has been reported is often derived from differentgenomic regions, making interfamily comparisons problematic; and (iii)the relative lack of sequence information on other host taxa has made itdifficult to distinguish between genuinely monophyletic HERV familiesand polyphyletic families that appear monophyletic only because similarviruses in other hosts have not yet been described. A pathogenicpotential of nondefective endogenous retroviruses has so far only beendemonstrated in mice, in which they may induce tumors and immunologicaldisorders. In human DNA, endogenous retrovirus sequences (HERVs) arewell known genetic elements. They all seem to be defective due tomultiple termination codons, deletions, or the lack of a 5′ longterminal repeat (LTR). Replication competent human endogenous retrovirusgenomes have not yet been isolated.

Extensive studies on animal melanomas (especially that of the hamster),which serve as model for human malignant melanoma have shown theoccurrence of virus particles in the tumors. A high molecular weight(70S) RNA species as well as an RNA-directed DNA polymerase, twodiagnostic features for oncorna (oncogenic RNA) viruses have beenassociated with these particles. Cell-free transmission experiments endto the conclusion that hamster melanomas are caused by an RNA virus.

Based on these findings the working hypothesis was proposed that humanmelanoma may also have a viral etiology. In experiments the occurrenceof virus-like particles as well as reverse transcriptase activity inmetastases of human melanoma was reported in the early 70s. (Birkmayeret al., Europ. J. Cancer 10 (1974) 419-422).

These virus-like particles are spherical or slightly ovoid and have adiameter of 90-120 nm. They have an electron-dense nucleoid measuring50-70 nm across and are bounded by a triple-layered, 100 Å thickmembrane. Sometimes fine projections extend radially from the nucleoidtowards the membrane, and in this case the particles resemble thosefound in hamster melanoma. Their number is rather small in comparison tothat found in hamster melanoma. So far, they have been seen only in thecytoplasm and no infectivity has been observed for these particles(Birkmayer et al., Die Naturwissenschaften 59 (8) (1972), 369-370).

Also Balda et al. (Proc. Nat. Acad. Sci, USA 72 (9) (1975, 3697-3700)described a high molecular weight RNA encapsulated with an RNA extractedDNA polymerase in particles possessing the identity characteristic ofthe RNA tumor viruses which was detected in human malignant melanomas.However, also here no infectious particles were observed.

Only many years later the expression of a family of human endogenousretrovirus sequences (HERV-K) in GH cells, a teratocarcinoma cell lineproducing the human teratocarcinoma-derived retrovirus (HTDV) particleswas described (Löwer et al., Proc. Natl. Acad. Sci. USA 90 (1993):4480-4484). Detailed electron microscopic surveys have revealed theexistence of retrovirus-like particles in human placentas andteratocarcinoma cell lines. However, these found HERV-K/HTDV particleswere also not infectious: They possess a distinct morphology: (i) theylack the electron lucent space normally seen between viral envelope andcore, (ii) they seem to be predominantly arrested in budding stages, and(iii) collapsed cores, a consequence of a final proteolytic step invirus maturation, have never been observed. As long as the gag precursorremains uncleaved, resulting particles are not likely to be infectious.

A further HERV-K provirus was shown to be present in gorilla andchimpanzee genomes but not in the human genome (Barbulescu et al.,Current Biology 11 (10) (2001), 779-783).

Therefore, human endogenous retroviruses have shown to be expressed,however, these virus particles found up to date were all non-infectiousvirus particles. Some endogenous retrovirus-like particles were found tobe expressed in melanoma cells. However, the only infectious endogenousretroviruses found were some animal endogenous retroviruses, e.g.MelARV, a retrovirus capable of infecting cultured murine melanocyteswhich have also been shown to induce in some instances malignanttransformation.

In the article by Reus et al. (Journal of Virology, October 2001,8917-8926) it is stated that the average age of HERV-K proviruses is ca.28 million years. It is described that at that time the HERV-Ks probablyused to be infectious.

The WO 01/62937 A1 relates to retrovirus sequences and psoriasis.

In the WO 00/53789 A1 various retroviral expression vectors are listed.

The WO 00/20460 A1 relates to the detection of a HERV-K10 gag-sequencewhich is brought into relationship with the cancer seminoma, whereby theHERV-KLO gag-sequence is used as tumor marker as well as for therapeuticmethods.

In the WO 00/06598 A1 a HERV-AVL3-B sequence fragment is brought inconnection with methods for the treatment for malign melanomas.

The WO 99/67395 A1 relates to a 600 bp sequence of an env-gene of avirus family HERV-7q and relates to the use for or in connection withmultiple sclerosis, and autoimmune diseases, respectively.

In the WO 98/24454 A1 the use of a retroviral ribozyme sequence HERV-PTNfor the inhibition of tumor growth is described.

The JP 9252780 A relates to a method, in which a retrovirus polypeptideof the type HERV-E as a surrogate marker for cancer and other illnessesis detected.

The WO 01/70941 A2 relates to a retroviral sequence with high homologyto the family HERV-H, whereby this sequence is brought into connectionwith multiple sclerosis.

The DE 198 11 692 A1 relates to a preparation for the protection againstsun rays which is effective against herpes simplex viruses, whereby thepreparation comprises organic and anorganic light filters.

The object of the present invention is therefore to provide at least afragment of a nucleotide sequence which codes for a further humanendogenous retrovirus which is infectious.

The object of the present invention is solved by a polynucleotidemolecule being a fragment of a nucleotide sequence of an infectioushuman endogenous retrovirus which polynucleotide molecule comprises asequence selected from the group consisting of

(a) a sequence with at least 98%, preferably 99%, still preferred 99.5%,identity to a sequence according to SEQ ID No 1,

(b) a sequence which hybridises under stringent conditions with anucleotide sequence according to said SEQ ID No 1,

(c) a sequence which differs from said sequences (a) or (b) due todegeneration of the genetic code and

(d) a sequence comprising sequences inserted into vectors pCR4-Topo anddeposited as “MERV-env”, “MERV-gag”, “MERV-prt” and “MERV-pol” at theDSMZ on 26 Sep. 2001 or fragments thereof.

It has been surprisingly found that a polynucleotide molecule asmentioned above codes for (parts of) an infectious human endogenousretrovirus. With the present invention it can be shown for the firsttime that not only infectious animal endogenous retroviruses exist butalso infectious human endogenous retroviruses. This is particularlysurprising due to the fact that human endogenous retroviruses have beenknown and described for more than 30 years, however, even though manygroups have been carrying out research in this field over decades,infectious human endogenous retroviruses could not be isolated ordetected so far.

The polynucleotide molecule according to the present invention ispreferably an isolated and a purified DNA or RNA molecule.

“Infectious” in the scope of the present application relates especiallyto retrovirus particles which are able to bind to the receptor of a cellover the env protein and enter into the cell and carry out a reversetranscription in order to produce a cDNA which can integrate into thehost cell DNA. The differences between the life cycles of differentretro elements and between infectious and non-infectious retroviruses isdescribed in the article by Lower et al. (Proc. Natl. Acad. Sci 93(1996), 5177-5184), which is incorporated herein by reference.

Preferably, “infectious” human endogenous retroviruses according to thepresent invention are able to infect bovine MDBK cells and to integrateinto these cells' genome.

The sequence according to SEQ ID NO 1 comprises a region which must bepresent in the polynucleotide molecule in order to code for aninfectious human endogenous retrovirus (HERV). However, at both ends thepolynucleotide molecule according to the pre-sent invention can comprisefurther herein not specifically described sequence regions, inparticular LTR regions common to such viruses. With the presentpolynucleotide molecule any here not defined sequence regions can beidentified by e.g. amplifying these sequences with the help of optimallydesigned gene specific primers which will for example bind to the abovedefined sequence regions. The amplification products can then besequenced. Such methods are well known to the person skilled in the art.

“Stringent conditions” relate to hybridisation reactions under definedhybridisation conditions which is a function of factors as concentrationof salt or formamide in the hybridisation buffer, the temperature of thehybridisation and the post-hybridisation wash conditions. Suchconditions for Northern Blot analysis are for example hybridisation at68° C. with a MERV-specific polynucleotide molecule labelled withdioxigenin or radioactive in a standard SSC hybridisation buffercontaining 0.1% SDS followed by stringent washing in wash buffer at thesame temperature. Stringent washing can be performed for example by twotimes washing with 2×SSC buffer followed by two wash steps with 0.5×SSCbuffer. In the present case stringent hybridisation conditions willpreferably involve a temperature of 15° C. to 25° C. below the meltingtemperature (Tm), whereby the Tm of a hybridisation product of a nucleicacid probe can be calculated using a formula based on the g+c containedin the nucleic acids and that takes chain lengths into account, such asthe formula Tm=81.5 to 16.6 (log [na⁺])+0.41 (% G+C)−600/N), whereinN=chain length (Sambrook et al. (1989), which is incorporated herein byreference). In practice an estimated Tm for an oligonucleotide probe isoften confirmed and thus a person skilled in the art can calculate theTm for any chosen probe whose nucleotide sequence is known.

Sequences which differ from a given sequence due to the degeneration ofthe genetic code can be defined by any person skilled in the art. Thiscan be carried out electronically without undue burden.

In the scope of the present application the deposited vectors MERV-env,MERV-gag, MERV-prt and MERV-pol refer to plasmids deposited at the DSMZ(Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) accordingto the Budapest Treaty and comprise the accession numbers DSM14540,DSM14538; DSM14536 and DSM14537.

In the scope of the present invention, when sequences inserted into thedeposited vectors are concerned not only identical sequences arereferred to but also sequences

(a) with at least 98%, preferably 99%, still preferred 99.5%, identityto the sequences inserted into the deposited vectors,

(b) which hybridise under stringent conditions with sequences insertedinto the deposited vectors and

(c) which differ from said sequences inserted into the deposited vectorsdue to degeneration of the genetic code.

The above polynucleotide molecule according to the present invention mayfurther comprise a sequence which is at least 99.7%, 99.8% or 99.9%identical to SEQ ID NO 1.

Preferably, the polynucleotide molecule comprises a sequence accordingto SEQ ID NO 1 and is therefore identical to SEQ ID NO 1. Thispolynucleotide molecule has shown to code for a complete and activeinfectious particle of a HERV. The ORFs of SEQ ID NO 1 are preferably asfollows:

ORF Nucleotides genetic code gag   1-2013 universal prt 1993-2809 nestedreading frame pol 3032-5650 universal env 5352-7451 universal

Still preferred a polynucleotide molecule is provided which comprises aspecific fragment of said above described polynucleotide molecule with alength of at least 15 bp, preferably at least 20 bp, still preferred atleast 30 bp. The term “specific” least 20 bp, still preferred at least30 bp. The term “specific” defines any fragment which comprises asequence different to any known HERV sequence. Therefore, any fragmentwith a length of at least 15 bp which comprises, for example, the inTable 1 mentioned mutations with respect to the known non-infectiousHERV K (GeneBank, accession nr. 164614) will be comprised by thepolynucleotide molecule according to the present invention. However,these given lengths are minimum lengths and also fragments which arelonger, e.g. 100, 1000, 1500 bp, are also comprised by the term“fragment”.

“Fragments” according to the present invention relate also to specificfragments of the sequences inserted into the vector pCR4-Topo anddeposited as MERV-env, MERV-gag, MERV-prt and MERV-pol as mentionedabove.

TABLE 1 position in HERV aminoacid K108 nucleotide change clone positionin (Accession change (MERV/ name MERV 164614) (MERV/HERV) HERV) MERV gag 416 1515 C/T Ala/Val  602 1701 C/A Arg/Lys  630 1729 A/G Pro/Pro  6391738 T/C Ala/Ala  654 1753 G/T Pro/Pro  738 1837 G/A Pro/Pro  799 1898G/A Glu/Lys  802 1901 C/T Leu/leu  866 1965 T/C Met/Thr  945 2044 A/GSer/Ser  953 2052 T/A Met/Lys  972 2071 A/G Glu/Glu 1149 2248 A/GGln/Arg 1348 2447 A/G Lys/Gly 1413 2512 C/T Ala/Ala 1642 2741 G/AGly/Arg 1653 2752 C/T Tyr/Tyr 1668 2767 C/T Ile/Ile 1701 2800 C/TAsn/Asn 1989 3088 A/G Pro/Pro MERV prot 2068 3167 T/C Ile/Thr 2087 3186C/A Gly/Gly 2090 3189 A/C Pro/Pro 2097 3196 G/A Glu/Lys 2100 3199 A/GArg/Gly 2124 3223 A/C Arg/Arg 2163 3262 G/A Gly/Ser 2198 3297 G/AGln/Gln 2238 3337 G/A Gly/Arg 2253 3352 G/C Glu/Gln 2273 3372 T/CTyr/Tyr 2305 3404 C/T Thr/Ile 2313 3412 T/C Phe/Leu 2330 3429 G/APro/Pro 2461 3560 G/A Arg/Gln 2501 3600 T/A Gly/Gly 2502 3601 T/CPhe/Leu 2543 3642 T/G Thr/Thr 2554 3653 G/A Arg/His 2590 3689 G/AArg/Gln 2654 3753 A/G Ala/Ala 2659-2661 3758-3760 —/ATC —/Ile 2677 3776T/C Leu/Ser 2741 3840 A/G Gly/Gly 2763 3862 G/A Val/Ile 2808 3907 T/ATyr/Asn 2815 3914 T/G Phe/Cys 2816 3915 T/C Phe/Cys MERV prot 2878 3977G/A Arg/Gln 2938 4037 G/A Arg/Gln 2974 4073 A/G His/Arg 3052 4151 C/TAla/Ala 3091 4190 T/C Pro/Pro 3105 4204 T/C Leu/Pro 3124 4223 C/TLeu/Asp 3178 4277 G/A Ala/Ala MERV pol 3363 4462 A/G Tyr/Cys 3664 4763A/C Ile/Ile 3700 4799 A/G Ile/Met 4040 5139 C/A Gln/Lys 4115 5214 A/TMet/Leu 4219 5318 G/A Gly/Gly 4225 5324 G/A Lys/Lys 4255 5354 T/GSer/Ser 4336 5435 C/A Ala/Ala 4465 5564 T/A Thr/Thr 4517 5616 G/CGlu/Gln 4771 5870 G/A Ser/Ser 4780 5879 T/C His/His MERV env 5402 6501C/T His/His 5719 6818 T/C Ile/Thr 6456 7555 G/A Val/Ile 6458 7557 C/TVal/Ile 6464 7563 A/G Leu/Leu 6802 7901 G/C Gly/Ala 7146 8245 A/GLys/Glu 7262 8361 A/G Thr/Thr 7340 8439 G/A Arg/Arg 7347 8446 A/GAsn/Asp 7388 8487 A/G Ser/Ser 7436 8535 C/T Val/Val

According to a preferred embodiment of the present invention apolynucleotide molecule encoding for an env protein of an infectiousendogenous human retrovirus is provided, said polynucleotide moleculecomprising a sequence selected from the group consisting of

(a) a sequence with at least 98%, preferably 99%, still preferred 99.5%,identity to a sequence according to SEQ ID No 2,

(b) a sequence which hybridises under stringent conditions with anucleotide sequence according to said SEQ ID No 2 and

(c) a sequence which differs from said sequence (a) or (b) due todegeneration of the genetic code.

“Env” according to the present invention relates also to the sequenceinserted into the deposited MERV-env plasmid as mentioned above or afragment thereof.

It is known that retroviruses are “quasispecies” and sequence variationin a given virus population is given. Such quasispecies variations are,of course, included in the definition of the present viruses and of thesequences of the nucleic acid molecules according to the presentinvention as long as the infectivity of the virus population as a wholeis given.

“Env” relates to the envelope protein which is a viral membrane proteinmediating the binding of the virus particles to the cellular receptorsenabling virus entry which is the first step in a new replication cycle.The env protein is particularly important for the ability to spreadbetween cells and individuals (infectivity).

A further embodiment of the present invention relates to apolynucleotide molecule encoding for a pol protein of a HERV, wherebysaid polynucleotide molecule comprises a sequence selected from thegroup consisting of

(a) a sequence with at least 99%, preferably 99.5%, still preferred99.8%, identity to a sequence according to SEQ ID No 3,

(b) a sequence which hybridises under stringent conditions with anucleotide sequence according to said SEQ ID No 3 and

(c) a sequence which differs from said sequence (a) or (b) due todegeneration of the genetic code. “pol” relates to the polymerase whichis an enzyme with RNA dependent reverse transcriptase activity. Alsothis protein is important for a correct viral replication cycle.

“Pol” according to the present invention relates also to the sequenceinserted into the deposited MERV-pol plasmid as mentioned above or afragment thereof.

A still further aspect of the present invention relates to apolynucleotide molecule encoding for a gag protein of a HERV, wherebysaid polynucleotide molecule comprises a sequence selected from thegroup consisting of

(a) a sequence with at least 98%, preferably 99%, still preferred 99.5%,identity to a sequence according to SEQ ID No 4,

(b) a sequence which hybridises under stringent conditions with anucleotide sequence according to said SEQ ID No 4 and

(c) a sequence which differs from said sequence (a) or (b) due todegeneration of the genetic code.

The gag gene products have acquired the ability to be transported to thecell surface and abut on the cell membrane incorporating env proteinsduring this process. The gag gene codes for a long polyprotein which ina first step is cut from the long molecule chain and is then cut into anumber of parts which are present around the RNA in form of capsomeres.Since these gag proteins are essential for the budding they areimportant for the infectious viral circle.

“Gag” according to the present invention relates also to the sequenceinserted into the deposited MERV-gag plasmid as mentioned above or afragment thereof.

A further embodiment relates to a polynucleotide molecule encoding for apro protein of a HERV, whereby said polynucleotide molecule comprises asequence selected from the group consisting of

(a) a sequence with at least 98%, preferably 99%, still preferred 99.5%,identity to a sequence according to SEQ ID No 5,

(b) a sequence which hybridises under stringent conditions with anucleotide sequence according to said SEQ ID No 5 and

(c) a sequence which differs from said sequence (a) or (b) due todegeneration of the genetic code.

The pro gene comprises 3 regions of particular interest:

1. dUTPase, dUTPase hydrolyses dUTP to dUMP and pyrophosphate.

2. prt retroviral Aspartyl-Proteinase

3. G_patch, α-glycine rich nucleic binding domain; A predicted glycinerich nucleic binding domain found in the splicing factor 45, SON DNAbinding protein and D-type Retrovirus-polyproteins.

“Pro” according to the present invention relates also to the sequenceinserted into the deposited MERV-prt plasmid as mentioned above or afragment thereof.

Advantageously, a polynucleotide molecule is provided which comprises asequence which is complementary to one of the above mentioned sequencesaccording to the present invention. Therefore, not only the sequenceaccording to the RNA strand present in the retrovirus particle isprovided but also a sequence according to the cDNA sequence.

Still preferred, the polynucleotide molecule comprises a detectablelabel. In particular if the polynucleotide molecule is a fragment of theabove defined sequence, the polynucleotide molecule can be used for thedetection of infectious HERV in a sample. The term “detectable label”relates to any marker well known in the art, e.g. fluorescent,radioactive, enzymatic or chemical marker.

A further aspect of the present invention relates to a polynucleotidemolecule coding for a ribozyme, which comprises two sections, each ofwhich has a length of at least 10 to 15 base pairs and which arecomplementary to specific sequence sections of said above identifiedpolynucleotide molecule according to the present invention so that saidribozyme complexes and cuts the mRNA transcribed by a natural infectioushuman endogenous retrovirus DNA. The publication by John M. Burg“Clearing the Way for Ribozymes” (Nat. Biotechnology 15:414-415 (1997))relates to the general mode of function of ribozymes and is includedherein by reference. The ribozyme will recognise the mRNA of infectiousHERV by complementary base pairing with said mRNA. The ribozyme willthen cleave and destroy the RNA in a sequence specific manner before theHERV is translated. After dissociation from the cleaved substrate theribozyme will repeatedly hybridise with specific RNA molecules and actas specific endonuclease. In general, ribozymes may specifically beproduced for inactivation of a certain mRNA even if not the entire DNAsequence which codes for the infectious HERV is known.

Ribozymes are particularly efficient if the ribosomes move slowly alongthe mRNA. In that case it is easier for the ribozyme to find a ribosomefree site on the mRNA. For this slow ribosomes mutants are also suitableas a system for ribozymes (J. Jayburg (1997), Nature Biotechnology414-415). A further possible way is also to use a varied form of aribozyme, i.e. a minizyme. Minizymes are efficient particularly forcleaving larger mRNA molecules. A minizyme is a hammer head ribozymewhich has a short oligonucleotide linker instead of the stem/loop II.Primer minizymes are particular efficient (Kuferbara et al. (1998),Nature Biotechnology 16, 961-965).

Here again the ribozyme comprises sequence sections which arecomplementary to “specific” sequence sections of the above definedpolynucleotide molecule according to the present invention. It issufficient if at least one of the two sequence sections is specific,meaning that this specific sequence section will only bind to aninfectious HERV according to the present invention and not to alreadyknown and described non-infectious HERV sequences. Preferably, however,both sequence sections are specific for the above defined polynucleotidemolecule according to the present invention, meaning that both sequencesections bind only infectious HERV sequences.

A further aspect of the present invention refers to a primer pairwhereby the primers specifically hybridise to a polynucleotide moleculeaccording to the present invention for an amplification reaction of afragment of said polynucleotide molecule. Here again the term“specifically” refers to primers which will only bind to the abovedefined polynucleotide molecule according to the present invention andnot to any known already described HERV. It is sufficient, if only oneof the primers is specific for the inventive polynucleotide molecule,however, preferably both primers bind specifically only to the inventiveHERV polynucleotide molecule. This primer pair can be used for anydetection reaction in order to detect the presence of a HERV in asample, whereby the term “amplification reaction” relates to anyreaction specifically amplifying a sequence corresponding to the abovedescribed HERV sequence according to the present invention. This may be,for example, a conventional PCR as well as RT-PCR which are both wellknown to a person skilled in the art. However, the primer pair can alsobe used to find further herein not defined parts of the infectious HERVprovirus according to the present invention. The primer pair canfurthermore be used to identify other infectious HERVs which comprise,at least partly, similar sequences to the sequence of the infectiousHERV according to the present invention.

The skilled man in the art easily finds conditions wherein primers whichare specific for the infectious HERV sequences according to the presentinvention (which e.g. differ from sequences of known (non-infectious)HERVs by only 1, 2 or 3 nucleotides) only hybridise or anneal to thesequences of the infectious HERVs according to the present invention,but not to e.g. the non-infectious HERV sequences known. Examples forsuch specific regions are e.g. the regions covering one or more of themutations listed in Table 1. Further examples may be derived by theskilled man in the art e.g. by comparing the sequences of the presentinfectious HERV with the known (non-infectious) HERV-K sequences (e.g.HERV-K108).

A further aspect of the present invention relates to a biologicallyfunctional vector comprising a polynucleotide molecule according to thepresent invention. The biologically functional vector will comprise allelements necessary for efficiently expressing the polynucleotidemolecule according to the present invention whereby these elements, e.g.the promoter will be selected according to the host into which thefunctional vector will be transfected and in which the polynucleotidemolecule will be expressed, e.g. to produce a protein or polypeptideencoded by the present infectious HERV sequences. A person skilled inthe art can select the optimal biologically functional vector withoutundue burden.

Still preferred, a biologically functional vector comprising apolynucleotide molecule as defined above according to the presentinvention in inverse orientation with respect to the promoter isprovided. This vector allows the production of antisense mRNA which iscomplementary to the mRNA of the infectious HERV according to thepresent invention. The antisense mRNA will bind to the respective regionof the infectious HERV mRNA and therefore block any further translationand production of viral proteins and therefore infectious viralparticles. Hereby it is sufficient, if an essential part of theinfectious HERV mRNA is blocked in order to block the production of acomplete infectious viral particle, e.g. part of the gag, pol, pro orenv mRNA. Therefore, said biologically functional vector can be used incell cultures to stop the viral production as well as a gene therapyagainst or prevention of viral replication in a patient.

A further aspect of the present invention relates to a recombinant hostcell stably transfected with said biologically functional vectoraccording to the present invention as defined above. Here, the personskilled in the art will select the vector and host cell in order toachieve an effective result, e.g. high expression of the infectious HERVor fragments thereof. Transfection methods in order to produce arecombinant host cell are well known to the person skilled in the artand may comprise any electroporation, lipofectamin transfection, salttransfection etc. Depending on the vector which is transfected into thehost cell, the host cell will either express viral peptides, proteins orparticles or produce antisense mRNA.

Still preferred the host cell is a mammalian cell. This will allow theproduction of infectious HERV proteins or peptides with correctpost-translational modifications. The mammalian cell may be, forexample, from any human cell line.

A further aspect of the present invention relates to a method forproducing an infectious human endogenous retrovirus or at least onefragment thereof comprising the steps of

(a) transfecting a host cell with a vector as defined above according tothe present invention,

(b) selecting and cultivating cells which are stably transfected withsaid vector and

(c) isolating and purifying said retrovirus or said fragment.

Here again the above mentioned definitions and preferred embodimentsapply, in particular with respect to the transfection step and theselection of the host cell and vector. The selection of stablytransfected cells may be carried out according to any method well knownin the art, e.g. selection with antibiotic resistance or opticalselection. The term “fragment” relates to any peptide encoded by theabove defined polynucleotide molecule according to the presentinvention. However, the fragment will preferably refer to a peptidewhich can be used as an antigen. To detect fragments which can be usedas antigens known homologous fragments of corresponding HERVs or ERVscan be used, however, such fragments can also be detected by providing asample of antibodies of infectious HERV, which sample can be taken of aninfected patient, incubating said antibody or antibodies with one ormore fragments of the infectious HERV proteins in conditions so that abinding between corresponding antigens and anti-bodies occur andidentifying the respective fragments which have bound to antibodies ofinfectious HERV.

A further aspect of the present invention relates to an infectious humanendogenous retrovirus comprising as RNA a polynucleotide moleculeaccording to the present invention. Preferably the infectious HERVaccording to the present invention will comprise the completepolynucleotide molecule, e.g. comprising the pol, env, gag, pro genes aswell as LTR sequences. However, it is also possible that the RNAcomprises only one or a few, but not all of the above mentioned genes.It is important that the HERV is able to carry out a complete retroviralreplication cycle and infect further cells.

Furthermore, an infectious human endogenous retrovirus is provided beingencoded by a polynucleotide molecule as defined above according to thepresent invention.

A further aspect of the present invention relates to a preparationcomprising an infectious HERV according to the present invention. Thepreparation may be a pharmaceutical preparation in which case preferablya pharmaceutically acceptable carrier is provided. However, thepreparation may also be designed for diagnostic purposes as well asanalytical or other purposes, e.g. to produce antibodies, to study theeffects in animals etc. Of course, the preparation according to thepresent invention will comprise any additional substances depending onthe use of the preparation. The preparation may comprise further virusparticles, as non-infectious HERVs. However, at least one infectiousHERV according to the present invention or a significant portion of thepreparation leading to the overall infectivity of this preparation mustbe present.

A further aspect of the present invention relates to an env protein, apol protein, a gag, a pro protein encoded by a polynucleotide moleculeas defined above according to the present invention. These proteins canbe used for any analytical, diagnostic or production methods and theywill be provided in any form, e.g. with additional substances, dependingon the use of the protein.

Furthermore a peptide is provided which is a specific fragment of saidretrovirus as defined above according to the present invention. Hereagain, “specific” refers to any fragment which is not identical to anon-infectious HERV fragment already known. As mentioned above alreadythe fragment may be any fragment of said retrovirus particle, however,preferably said fragment can be used as an antigen (e.g. a T or B cellepitope) and therefore be used in diagnostic methods as well as for theinduction of the production of respective antibodies in a patient oranimal and/or for the induction of specific T cell responses, both TH1and TH2. The detection of such fragments can be carried out as describedabove.

The following fragments are examples of such antigens:

Fragments of the gag protein may be:

L M Q N E A I E Q V R A I C L (SEQ ID NO:9), I P Y D W E I L A K S S L SP (SEQ ID NO:10), A D Q L L G I G Q N W S T I S (SEQ ID NO:11), T I S QQ A L M Q N E A I E Q (SEQ ID NO:12), E K A R K V I V E L M A Y E N (SEQID NO:13), M A Y E N A N P E C Q S A I K (SEQ ID NO:14), P V L N K Q N IT I Q A T T T (SEQ ID NO:15), R S K F D K N G Q P L S G N E (SEQ IDNO:16), L S G N E Q R G Q P Q A P Q Q (SEQ ID NO:17), Q P P L S Q V F QG I S Q L P (SEQ ID NO:18), E I I D K S R K E G D T E A W (SEQ IDNO:19), V S T K N L I K L (SEQ ID NO:20), G I G Q N W S T I (SEQ IDNO:21), Q Y G P N S P Y M (SEQ ID NO:22), C P V L N K Q N I (SEQ IDNO:23), L T V W N D W A I (SEQ ID NO:24), K F D K N G Q P L (SEQ IDNO:25), G K C Y N C G Q I (SEQ ID NO:26), H L K K N C P V L (SEQ IDNO:27), G R K G N I I P L (SEQ ID NO:28), F S I K M L K D M (SEQ IDNO:29)

Fragments of the env protein may be:

P A V D S D L T E S L D K H K (SEQ ID NO:30), W N S Q S S I D Q K L A NQ I (SEQ ID NO:31), V S M D R P W E A S P S V H I (SEQ ID NO:32), P A VQ N W L V E V P T V S P (SEQ ID NO:33), L R P R V N Y L Q D F S Y Q R(SEQ ID NO:34), N T E V L V W E E C V A N S A (SEQ ID NO:35), S A V I LQ N N E F G T I I D (SEQ ID NO:36), Q F Y H N C S G Q T Q S C P S (SEQID NO:37), N R S K R F I F T L I A V I M (SEQ ID NO:38), P Y M L V V G NI (SEQ ID NO:39), I F K A S K A H L (SEQ ID NO:40), K T I G S T T I I(SEQ ID NO:41), G Y H Y P P I C L (SEQ ID NO:42), S Y Q R S L K F R (SEQID NO:43), K G K P C P K E I (SEQ ID NO:44), V E V P T V S P I (SEQ IDNO:45), S L R P R V N Y L (SEQ ID NO:46), T F N W Q H R I L (SEQ IDNO:47)

Preferably, such peptide fragments contain at least 6 amino acidresidues with at least one residue being specific for the pre-sentinfectious HERV sequences, especially at least one of the amino acidresidues not being present in the (non-infectious) HERV-K (HERV-K108).More preferred these peptides have a length from 7 to 15 amino acids,especially from 8 to 11. Such peptide fragments of these lengths arepreferably fragments of the above mentioned gag and env fragments.

A further aspect of the present invention relates to an antibody whichis directed against an antigen derived from said retrovirus as definedabove according to the present invention. The production of antibodiestowards any specific antigen may be carried out according to any methodwell known in the state of the art, e.g. by immunisation of an animaland producing the antibodies in cell culture. The term “antibody” refersto any form of antibody, e.g. monoclonal, polyclonal, humanized antibodyetc. The antibodies directed against antigens from the infectious HERVaccording to the present invention can be used in immunizing methods,diagnostic methods or therapeutic methods.

Preferably, an antibody fragment is provided which is directed againstan antigen derived from said retrovirus as defined above according tothe present invention. Such antibody fragments may be for example Fa,F(ab)² or Fv which are capable of binding an epitopic determinant.Furthermore, single chain antibodies are comprised by the term “antibodyfragment”. These antibody fragments can be used as described above forthe antibodies, however, due to their smaller size they may showadvantages with respect to stability, production etc. Also here, theantibody fragment is specific, meaning that the antibody fragmentcomprises a sequence specific to infectious HERV according to thepresent invention.

A further aspect of the present invention relates to a solid supportwhich has attached to its surface said antibodies defined aboveaccording to the present invention or said antibody fragments as definedabove according to the present invention. The solid support according tothe present invention may be of any size or shape, e.g. in the form of aflat slide, microtiter plate, chip, filter, column, membrane etc. andmay be made of any material generally used for solid supports, e.g.modified or unmodified glass, polymeric material etc. Hereby theantibody or antibody fragment can be attached to the complete surface ofthe solid support. However, it may also be attached in localisedregions, as for example spots. Also the quantity in which the antibodyis bound to said solid support may vary according to the size of theantibody or antibody fragment. Of course, for high throughput assays itwill be advantageous to use micro array chips on which the antibodies orantibody fragments are bound in high density. The selection of the solidsupport and the mode of attachment of the antibody to its surface willdepend on the use of said solid support, e.g. for preparation,purification methods, detection methods, etc. and a person skilled inthe art will be able to select the most appropriate solid support andthe optimum mode of attachment of said antibody or antibody fragment tosaid solid support.

A further aspect of the present invention relates to a method fordiagnosing cancerous cells comprising the steps of

(a) providing a sample of said cells to be tested or supernatantthereof,

(b) analysing whether or not said infectious endogenous retrovirusaccording to the present invention as described above or fragmentthereof is present in said sample whereby

(c) the presence of said retrovirus or fragment thereof in said samplediagnoses cancerous cells.

The term “cancerous cells” refers to malignant tumor cells. It has beensurprisingly found that the infectious HERV according to the presentinvention is related to the transformation of cells into cancerouscells, especially melanomas. Therefore, by detecting the presence ofsaid infectious HERV it is possible to diagnose cancerous cells alreadyat a very early stage. Therefore, the term “diagnosing cancerous cells”includes also diagnosing cells which will be transformed into cancerouscells, however, with conventional methods these cells would not yet bediagnosed as transformed.

However, it is not necessary to detect the complete infectiousendogenous retrovirus. It is sufficient to detect at least one specificfragment thereof. Here again, the term “specific” refers to a fragmentcontaining a sequence which differs from the sequences of the knownnon-infectious HERVs. The detection method may be carried out accordingto any protocol known in the art whereby it is possible to detect saidinfectious HERV or fragment thereof on DNA, RNA or protein level. It isfurther possible to determine the stage of cancer by determining theamount of infectious HERV.

Preferably, said analysing step comprises an antigen-antibody reactionusing an antibody or antibody fragment as defined above according to thepresent invention in order to detect proteins or protein fragments ofsaid retrovirus. Here, any known antigen-antibody reaction may becarried out, e.g. on solid support, in liquid phase, by sandwich-ELISA,etc. It is, of course, possible to detect antibodies in said sample,however, since the production of antibodies may vary, it is preferred todetect an antigen in said sample by providing one or more specificanti-bodies whereby these antibodies may be bound to a solid supportand/or comprise a label which may be detected and quantified.

According to a preferred method said analysing step comprises detectingthe polynucleotide sequence of said retrovirus and a fragment of thepolynucleotide sequence specific for said retrovirus, respectively,whereby a hybridisation reaction or amplification reaction is carriedout using a polynucleotide as defined above according to the presentinvention. Hereby, the term “polynucleotide sequence” comprises RNAsequences, e.g. the polynucleotide molecule derived from thetranscription of the respective DNA or the polynucleotide moleculepresent in the virus particle, as well as DNA, e.g. cDNA resulting fromthe reverse transcriptase reaction thereafter, whereby the cDNA may bestill present in the virus particle or also the DNA already integratedinto the host DNA. Therefore, said amplification reaction will comprisePCR as well as RT-PCR. Therefore, the above mentioned primer pair willpreferably be used.

Advantageously, said polynucleotide sequence is transformed into cDNA ina reverse transcription step prior to said hybridisation step andamplification reaction, respectively. Said hybridisation step will bepreferably carried out with one of the above defined fragments of saidpolynucleotide molecule according to the present invention as mentionedabove. Hereby it is, of course, possible to use labelled fragments orlabelled nucleotides, e.g. retroactive nucleotides in order to easilydetect and quantify, respectively, said polynucleotide sequence.

According to a further preferred method said analysing step comprises

(a) providing cells devoid of said endogenous retrovirus,

(b) adding said supernatant of said sample to said cells,

(c) cultivating said cells and

(d) detecting a polynucleotide molecule as defined above according tothe present invention in the DNA of said cells.

Since the HERV according to the present invention is infectious, thesupernatant of said sample—if it comprises an infectious HERV—willinfect the cells devoid of said endogenous retrovirus. After infectionthe infectious HERVs will integrate into said cells and further HERVparticles will be produced. After cultivating said cells, therefore, theintegrated polynucleotide molecule of the infectious HERV as definedabove will be detectable in the DNA of said cells. This is a veryreliable and quick method for detecting infectious HERVs. Again, thedetection of said polynucleotide molecule may be carried out by ahybridisation step and amplification reaction, respectively.

Preferably said cells are non-human cells. Since non-human cells do notcomprise HERVs, the detection of a HERV polynucleotide molecule in saidhuman cells will be a clear method of detecting infectious HERVparticles. Preferably, said non-human cells are bovine cells.Infectivity of the virus particles with respect to these non-human cellsis also a way of defining infectivity according to the presentinvention.

Still preferred said sample is a skin tissue sample from a patient.Since said infectious HERVs preferably infect skin tissue cells, asample of a skin tissue is preferably used in order to detect saidinfectious HERVs.

Still preferred, said sample comprises melanocytes. Since saidinfectious HERVs are related to cancerous cells a sample comprisingmelanocytes is a preferred sample for detecting infectious HERVsaccording to the present invention.

According to a preferred embodiment of the present invention a method isprovided for diagnosing cancer in a patient comprising the steps of

(a) providing a sample of said patient to be tested,

(b) analysing whether or not at least one antibody as defined above orantibody fragment as defined above according to the present invention ispresent in said sample whereby

(c) the presence of said antibody or fragment thereof in said samplediagnoses cancer.

Here again, the same definitions and preferred embodiments as mentionedabove apply.

Preferably said sample is a blood sample of said patient. A blood sampleis very easily and quickly taken from a patient without necessity ofcostly apparatuses. Furthermore, it is possible to diagnose an unlimitedamount of samples in a very short time. As mentioned above for highthroughput assays it is preferable to use microarrays.

According to an advantageous method said analysing step comprises anantigen-antibody reaction using an antigen derived from said retrovirusas defined above according to the present invention. Here again, theabove mentioned definitions and preferred embodiments apply.

According to a further advantageous embodiment a kit is provided fordiagnosing cancerous cells according to the method as mentioned aboveaccording to the present invention comprising

(a) a first reagent comprising a polynucleotide molecule as definedabove according to the present invention, an antibody as defined aboveaccording to the present invention or an antibody fragment as definedabove according to the present invention or a solid support as definedabove according to the present invention and

(b) as a positive control a second reagent comprising a polynucleotidecomprising a sequence which is complementary to the sequence of saidpolynucleotide in said first reagent or a retrovirus as defined aboveaccording to the present invention, an env protein as defined aboveaccording to the present invention, a pol protein as defined above, agag protein as defined above or a peptide as defined above according tothe present invention. Here again, the same definitions and preferredembodiments as described above apply. Of course, it is also possible toprovide additionally to the first reagent a further solid supportwithout attached antibodies or antibody fragments. In this case theantibody or antibody fragment as well as the mode of attachment can bevaried.

According to a further embodiment of the present invention a kit forcarrying out a method for diagnosing cancer in a patient as describedabove is provided comprising

(a) a first reagent comprising an antigen derived from said retrovirusas defined above and

(b) as a positive control a second reagent comprising an anti-bodyaccording to the present invention or an antibody fragment as definedabove specific for said antigen in said first reagent. Here again thesame definitions and preferred embodiments as described above apply.

Preferably, said antigen is labelled with a detectable marker. Thismarker may be for example radioactive, fluorescent, an enzymatically orchemically detectable marker. Such markers are well known in the stateof the art and the person skilled in the art will be able to select theoptimal marker.

Still preferred, said antigen is immobilised to a solid support. Thisallows an easy handling of said kit and the method can be carried outimmediately without prior preparations.

According to a further embodiment of the present invention a PNAmolecule is provided which comprises a base sequence complementary tothe sequence of said polynucleotide molecule as defined above accordingto the present invention. PNA (peptide nucleic acid) is a DNA likesequence, the nucleic basis being bound to a pseudo peptide backbone.PNA generally hybridises with complementary DNA, RNA or DNA polymers byWatson-Creek base pair and helix formation. The peptide backbone ensuresa greater resistance to enzymatic degradation. The PNA molecule thus isan improved antisense agent.

Here, the advantage lies in that neither nucleases nor proteases arecapable of attacking a PNA molecule. Therefore, the PNA molecule ishighly stable, if it is bound to a complementary sequence, since itcomprises a sufficient steric blocking of DNA and RNA polymerases,reverse transcriptase, telemorase and ribosomes (Buga et al, “CellPenetrating DNA Constructs Regulate Alanin Receptor Levels and ModifyTransmission in vivo”, Nature Biotechnology 16:857-861 (1998)).

As in the case of the antisense RNA, if the PNA molecule comprises theabove defined sequence it will bind to the DNA/RNA or to a site of theDNA/RNA respectively, which codes for infectious HERV peptides. In thisway, the PNA molecule will inhibit the transcription of the virus. ThePNA molecule is prepared synthetically, e.g. by aid of the T-boctechnique, since it is neither transcribed nor translated.

The present invention also relates to the use of anti-retroviralsubstances for preparing a medicament to treat melanoma. The presentinvention therefore provides a method for treating patients having orbeing at risk of developing melanoma comprising administering aneffective amount of substances acting against retroviruses, e.g.substances which inhibit replication and polymerisation of retroviral(infectious HERV) genomes. Examples of such treatment are e.g. known forthe treatment of HIV infections.

Preferably, a pharmaceutical composition is provided comprising apolynucleotide as defined above according to the present invention, avector as defined above according to the present invention, an antibodyas defined above, an antibody fragment as defined above and a PNAmolecule as defined above, respectively, and a pharmaceuticallyacceptable carrier. Of course, the pharmaceutical composition maycomprise any further molecules or substances useful for a therapeuticalmethod. The pharmaceutical composition may for example be provided as askin cream or lotion, which may for example be used as an after sunlotion in a preventive manner.

Preferably, the pharmaceutical composition further comprises at leastone antiviral substance, especially anti-retroviral substances. Thesemay be any substances well known in the art, for example the agents usedin known triple anti-retroviral therapy, see for example Bartlett J A,DeMasi R, Quinn J, Moxham C, Rousseau F. Overview of the effectivenessof triple combination therapy in antiretroviral-naive HIV-1 infectedadults. AIDS. 2001 Jul. 27; 15(11):1369-77; Marimoutou C, Chene G,Mercie P, Neau D, Farbos S, Morlat P, Ceccaldi J, Dabis F. Prognosticfactors of combined viral load and CD4+ cell count responses undertriple antiretroviral therapy, Aquitaine cohort, 1996-1998. J AcquirImmune Defic Syndr. 2001 Jun. 1; 27(2):161-7, which are incorporatedherein by reference.

According to a further embodiment of the present invention a sun creamis provided which comprises a polynucleotide as defined above accordingto the present invention, an antibody, an anti-body fragment as definedabove and a PNA molecule as defined above, respectively, and at leastone UV-protection substance. Therefore, applying a sun cream with thesesubstances will pre-vent or inhibit the production of or infection byinfectious HERV according to the present invention.

Preferably, said sun cream further comprises at least one anti-viralsubstance. Here, the same antiviral substance as mentioned above for thepharmaceutical composition will be preferred.

According to a further embodiment of the present invention a vaccine isprovided which comprises as an antigen an attenuated retrovirus asmentioned above, an env protein, a pol protein, a gag protein or apeptide as defined above according to the pre-sent invention,respectively, and optionally an adjuvant. Such a vaccine when applied toa patient will induce the production of antibodies and/or T cells, whichcan inhibit infectious HERVs according to the present invention.Therefore, such a vaccine is a highly effective substance for treatinginfectious HERV infections. The production of vaccines againstretroviruses is described in “Hyperattenuated Recombinant Influenza AVirus Nonstructural-Protein-Encoding Vectors Induce HumanImmunodeficiency Virus Type 1 Nef-Specific Systemic and Mucosal ImmuneResponses in Mice”, Boris Ferko, Jana Stasakova, Sabine Sereinig, JuliaRomanova, Dietmar Katinger, Brigitte Niebler, Hermann Katinger, andAndrej Egorov. Journal of Virology, October 2001, p. 8899-8908, Vol. 75,No. 19, which is incorporated herein by reference.

The present invention is described in more detail with the help of thefollowing examples and figures to which it should, however, not belimited.

In the drawings:

FIG. 1 shows the characterisation of particle preparations from melanomacell supernatants.

FIG. 2 shows an electron microscopy of melanoma cell-derivedsupernatants.

FIG. 3 shows the expression of the retroviral pol gene in melanomas.

FIGS. 4A and 4B show an immunofluorescence analysis with HERV-K-specificantibodies against gag, cORF, and env.

FIG. 5 shows cells infected with melanoma-associated particles.

FIG. 6 shows the detection of particle associated RNA and supernatantsfrom infected MDBK cells.

FIGS. 7A and 7B show the transfer of supernatants from melanoma particleexposed MDBK cells to uninfected MDBK cells.

FIGS. 8A and 8B show the vector pCR4Topo (SEQ ID NOs:48 and 49) as wellas the gag (SEQ ID NO:50), prot (SEQ ID NO:51), pol (SEQ ID NO:52) andenv (SEQ ID NO:53) primers.

EXAMPLES Example 1 Human Melanoma Cells Contain RT Activity in theirSupernatants

To detect extracellular virions, supernatants from the melanoma celllines SKMel-28, SKMel-1, 518A2 were analysed, as well as primarymelanoma cells for pelletable RT. Supernatants from cultured humanneonatal melanocytes (NHEM) served as controls. Cell-free supernatantsfrom approximately 10⁶ cells were concentrated by centrifugation. The RTactivity of the resulting pellets was analysed by F-PERT assay.

Supernatants from melanoma cell lines, and normal human melanocytes werecentrifuged at 3000×g at 4° C. and sterile-filtered through a 0.22 μmlow-protein membrane (Nunc) to remove cells and cellular debris. Theclarified supernatants were centrifuged for 20 min at 250000×g at 4° C.in a Beckman SW50.1 rotor. Pellets were rinsed with phosphate-bufferedsaline (PBS) and centrifuged for 15 min at 250000×g and resuspended. Theresulting particle suspensions were used as an enzyme source for RTassays.

Pelleted particles derived from 4 ml cell-free supernatants ofapproximately 10⁶ cells were analysed. RT activity was determined byfluorescent probe-based product enhanced RT (F-PERT) assay as described,with modifications. First, pellets were suspended in lysis buffer(Roche). Then RT was performed, using the suspended pellet as source ofenzyme, the primer 3′A10 (5′-CACAGGTCAAACCGCCTAGGAATG-3′ (SEQ IDNO:54)), and 0.3 μg MS2-RNA as a template (Roche # 165948). To limitunspecific reverse transcription, 0.5 μg calf-thymus DNA (Sigma # D4522)was added. After incubation at 42° C. for 1 h, a 5 μl aliquot of thisreaction was amplified by real-time PCR, by adding 25 μl TaqManUniversal PCR Master Mix (Applied Biosystems # 4304437), 1 μl of theprimers 3′A10 and 5′A 11 (5′-TCCTGCTCAACTTCCTGTCGAG-3′ (SEQ ID NO:55))at a concentration of 10 μM each, 1 μl F-PERT probe (genxpress, 10 μM,5′(FAM)-TCTTTAGCGAGACGCTACCATGGCTA-(TAMRA)3′ (SEQ ID NO:56)), and 17 μlAD. The resulting mixture was then amplified by incubating 10 mins at95° C., followed by 40 cycles at 94° C. for 20 sec and 64° C. for 1 min,in a SD 7700 (Perkin Elmer). The calibration curve was generated byplotting the RT activity of a serial dilution of Moloney Murine LeukemiaVirus (M-MLV) RT (Superscript II Gibco # 18064-014). Alternatively,detection of reverse transcriptase was performed with a commerciallyavailable reverse transcription assay (Boehringer Mannheim). In thisprocedure, dioxigenin (DIG) and biotin-labelled nucleotides synthesisedby the reverse transcriptase are incorporated into the DNA molecule.Biotion-labelled DNA is then captured to the streptavidin coated surfaceof the microtiter plates. In a subsequent step, an antibody to DIGconjugated to peroxidase is bound to the dioxigenin-labelled DNA. In thefinal step, the peroxidase substrate ABTS and a substrate enhancer wereadded. The absorbance of the samples was determined using a microtiterplate reader. The calibration curve was generated by plotting a serialdilution of an HIV-1 derived RT with defined activity.

Supernatants were found to contain a fluctuating yet continuouslypresent RT activity, corresponding to the activity of up to 10000 μunitsof M-MLV RT (Superscript II, Gibco) per ml supernatant. On the otherhand, supernatants from cultured melanocytes did not contain detectableRT activity (detection limit 10⁻¹ μunits). The fact that supernatantsderived from melanoma cells but not from melanocytes contain pelletableRT activity shows that melanoma cells contain proviral sequences withsufficient genetic information to form particles containing a functionalRT. Since no RT activity was detected in the supernatants ofmelanocytes, production of particles containing RT appears to beactivated during transformation of melanocytes to malignant cells.

Example 2 Human Melanoma Cell Derived Particles Package Sequences withHigh Homology to HERV-K

Retroviral pol genes are generally the most conserved sequences amongretroviruses. In an attempt to analyse the particle preparations on amolecular level, a sequence was amplified by using degenerate primerscorresponding to conserved sequences of the pol gene. Particlepreparations of the melanoma cell line 518A2 and SK-Mel28 were used forRT-PCR analysis. Particles were treated with DNAse, to removecontaminating traces of genomic DNA.

Oligonucleotide primers 3′ABDPOLS

5′dCATTCCTTGTGGTAAAACTTTCCA[T/C]TG 3′ (SEQ ID NO:57) and

5′ABDPOLAS 5′dCCCCTTGGAATACTCCTGTTTT[T/C]TG 3′ (SEQ ID NO:58) (CodonGenetic Systems, Austria), derived from conserved regions within theretroviral reverse transcriptase (RT), were adopted from Medstrand andBlomberg. In order to release viral RNA, the particle suspension wastransferred to a 0.5 ml polypropylene microcentrifuge tube (SörensenInc., USA). Then 10 μl of sterile water containing 0.1% Triton X-100 and10 U RNase inhibitor (Gibco) was added and incubated at 65° C. for 10min. The reverse transcription was performed in the same tubes. Amixture containing 50 mM Tris-HCl buffer pH 8.3, 50 mM KCl, 7.5 mMMgCl₂, 5 mM DTT, 1 mM dNTPs, 20 μmol primer 3′ABDPOLA, 10 U RNaseinhibitor and 100 U of Superscript II RT (Gibco) in a final volume of 20μl was added while the tubes were kept on ice. The reaction wasperformed at 42° C. for 45 min.

For PCR, the reaction mixture contained 10 mM Tris-HCl buffer pH 8.3, 50mM KCl, 1.5 mM MgCl₂, 0.005% (w/v) gelatin, 0.2 mM dNTPs, 25 μmol of3′ABDPOLS and 5′ABDPOLAS, 10 μl of the reverse transcription reactionand 1.25 U Taq polymerase (Boehringer Mannheim) in a final volume of 50μl. The reaction mixes were overlaid with mineral oil and subjected to 3thermal cycles of denaturation at 92° C. for 45 s, primer annealing at45° C. for 45 s and elongation at 72° C. for 60 s, followed by 45 cyclesat 92° C. for 45 s, 55° C. for 45 s and 72° C. for 60 s. 10 μl of eachamplification product were analysed by electrophoresis in a 1.5% agarosegel and visualised by ethidium bromide staining. An aliquot of theamplification product was cloned and sequenced.

RT-PCR of these preparations revealed the anticipated amplificationproducts of 298 nucleotides. Since no amplification products wereobtained when RT was omitted, possible DNA contaminations can beexcluded (FIG. 1 a). (A) RT-PCR. Particle preparations of the melanomacell line Mel-Juso were treated with DNAse and used for analysis. PCR(lane 1) or RT-PCR (lane 2) with degenerate primers corresponding tosequences that are conserved in retroviral polymerase (pol) genes wasperformed; M, molecular weight marker (Boehringer Mannheim VIII). Noamplification products were obtained in the control reactions withoutRT, demonstrating the absence of contamination with DNA (lane 1). Incontrast, RT-PCR revealed the anticipated amplification products of 298nucleotides (lane 2). (B) Immunoblotting. Particle preparations fromSK-Mel28 supernatants were separated by SDS-PAGE, blotted andHERV-K-specific proteins were detected with antiserum recognising env(lane 1), gag (lane 3) and the corresponding pre-immunesera, pre-env(lane 2) and pre-gag (lane 4). Molecular weight is indicated to theleft. In addition, RT-PCR of the GAPDH gene was negative, reflecting thelack of contamination with cellular mRNA (data not shown). Theamplification product was cloned into an expression vector andsequenced. Sequence analysis revealed that the amplified fragment showshigh homology to the corresponding pol region of endogenous retrovirusHERV-K. Translation of the obtained nucleotide sequence revealed theamino acid sequenceIKKKSGKWRMLTDLRAINSVIQPMGALQPGLPSPAIIPKNWPLVVIDLKDSFFTIPLADQDCEWFAFIIPAVNNLQPAKHF (SEQ ID NO:59). This sequence is highly homologouswith the corresponding sequences of the HERV-K HML2 and HML3 families.Within these groups the highest identity (90%) was observed for cloneNMWV5 (GenBank accession AF0115998). Homology to sequences of type A, Band C retroviruses was less than 70%. The sequence analysis excludescontamination with any known non-human retrovirus and suggests anendogenous origin of the packaged RNA.

Example 3

Human melanoma derived particles contain mature gag and env proteinsParticles derived from SK-Mel28 melanoma cells were purified oniodixanol-cushions and analysed for the presence of HERV-K-specific envand gag proteins in Western blots.

Iodixanol-cushion purified particles from SK-Mel28 supernatants werepelleted, resuspended in PBS and analysed. The amount of solubleproteins was quantified by means of a modified Bradford analysis(Bio-rad, Richmond Calif.). 5 μg of total protein was applied per laneand separated by SDS-PAGE (10%). Proteins were transferred to PVDFmembranes (Millipore, Bedford, Mass.) by Western blotting, and generatedblots were incubated with the gag- and env-specific antisera. Themembranes were washed twice with blocking solution and HERV-K-specificproteins were detected with an alkaline-phosphatase-conjugatedsecond-step antibody.

The envelope (env) gene of retroviruses displays an ORF for the surfaceprotein (SU) and a membrane spanning protein (TM). The env precursor isusually cleaved into the SU and TM subunits before translocation to thecell surface and incorporation into virus particles. To determinewhether the env protein is present on the particles, immunoblotting withan antiserum recognising the TM domain was performed. As shown in FIG. 1b two bands are visible in the Western blot. The upper band correspondsto the precursor migrating at approximately 80 to 90 kDa. In addition, alower band migrating at approximately 37 kDa is visible, suggestingcleavage of the precursor into subunits.

Immunoblotting with an HERV-K anti-gag antiserum revealed a double bandat approximately 76 kDa, corresponding to gag precursors, as well asprocessed intermediate gag proteins at approximately 61 kDa, 30 kDa andone band migrating with the front (less than 19 kDa). The 30 kDa proteincorresponds to the putative major core protein of HERV-K. The presenceof processed gag proteins in the Western blots indicates a functionalprotease.

Example 4 Human Melanoma Cells Produce Retrovirus-Like Particles

To confirm the presence of particles electron microscopy (EM) wasperformed.

15 μl aliquots of iodixanol-density gradient purified particlesuspensions from SK-Mel28 supernatants were applied on Formvar coatedgrids and left there for 15 min. Excess suspension was removed from theedges of the grids by filter paper. The grid with the remaining samplewas air dried for 1 h. The sample was either directly exposed to 1%uranyl acetate for negative staining or, for immunoelectron microscopy,fixed in paraformaldehyde-lysine-periodate for 15 min, rinsed indistilled water, quenched in PBS/1% BSA and incubated in anti-envantiserum diluted 1:25 in PBS/1% BSA for 1 h at RT. After washing, thegrid was exposed to 5 nM colloidal gold conjugated rabbit anti-goatantibody (British Biocell, Cardiff, UK) diluted 1:50 in PBS/1% BSA for 1h. After fixation in 2.5% glutaraldehyde, washing and negative stainingwith 1% uranyl acetate, the grid was left to air dry. All grids wereexamined with a JEOL 1010 electron microscope.

For electron microscopy, immunoblotting and infection studies, cell-freesupernatants were purified on iodixanol density gradients or cushions.Iodixanol is an iodinated, nonionic density gradient medium (NycomedPharma, Oslo, Norway). It has a low viscosity and provides iso-osmoticconditions up to densities of 1.32 g/ml. The supernatants were overlaidon a cushion of 5 ml 50% iodixanol. The tubes were centrifuged in anSW28 rotor at 45000×g for 2 h at 4° C. and the supernatant was removedfrom the tubes by suction, leaving a volume of 4 ml of the medium inproximity of the cushion. This fraction was harvested, pelleted in aSW41 rotor at 150000×g for 90 min at 4° C., resuspended in PBS andanalysed. Alternatively, for further purification the cushion-derivedfraction was loaded on iodixanol density gradients, and the fractioncorresponding to a density of approximately 1.16 g/ml was harvested. Theharvested fractions were diluted in PBS and pelleted in a SW41 rotor at150000×g for 90 min at 4° C. and resuspended in PBS.

Pellets were partially purified on iodixanol-cushions (FIG. 2, upperpart). Alternatively, these fractions were further purified on iodixanoldensity-gradients (FIG. 2, lower part). These preparations were analysedby immunelectron microscopy using an env-specific antiserum and acolloidal gold conjugated reporter antibody. EM analysis of thesepreparations revealed the presence of retrovirus-like particlescharacterised by membrane bound spherical structures with diametersranging between 80 and bound spherical structures with diameters rangingbetween 80 and 120 nm. The presence of immunogold on the particlesindicates that the env protein is present in the envelope.

Example 5 The Pol Gene is Expressed in Tumor Cells Derived fromMelanomas

The integration sites of human endogenous retroviral elements have beenfound to be distributed over the whole human genome. For example, theHERV-K family was reported to be present in approximately 30 copies perhuman haploid genome. Based on the observation that melanoma cells (butnot melanocytes) produce retrovirus-like particles, it can behypothesized that the formation of virus-like particles might be due tothe activation of retroviral genes that are usually repressed. Thereforethe cloned pol sequence derived from the particle preparations was usedas a probe and looked for expression of this sequence in the cytoplasmof melanoma cells by in situ hybridisation.

Touch preparations of a nevus, primary melanoma, and melanoma metastasiswere made by dipping freshly excised tissue on coated slides (DAKO,Biotek Solutions). Slides were fixed in 4% paraformaldehyde for 20 mins,washed in PBS, dehydrated through graded alcohols to absolute ethanol,and were air dried. The hybridisation mixtures consisted of Hybrisol VI(Oncor, Gaithersburg) and DIG-labelled cDNA probes, giving a finalconcentration of 2 ng/μl. Slides were covered with glass coverslips andsealed with Gelbond (ICN). Probe and cellular material were denatured byheating to 80° C. for 5 mins. Hybridisation was carried out at 37° C.overnight in a humid chamber. After removal of the coverslips the slideswere washed at 46° C. three times with 50% formamide/2×SSC, once with2×SSC for 10 mins, followed by a single wash step in 2×SSC containing 0,1% NP40 for 10 mins. Signal detection was performed after a blockingstep in 1% blocking reagent (Boehringer Mannheim) of 30 mins at 37° C.by incubation with anti-DIG antibody, conjugated to rhodamine at adilution of 1:10 in 1% blocking reagent for 30 mins at 37° C. in ahumidified box. After extensive washings in PBS, slides werecounterstained with 10 μg/ml DAPI for 20 mins and visualised with aZeiss fluorescence microscope using a triple bandpass filter andsoftware from PSI.

To determine the specificity of the pol sequence for melanoma, touchpreparations of a nodular melanoma were analysed, a lymph nodemetastasis, and a cutaneous metastasis that had been surgically removedfrom melanoma patients. As shown in FIG. 3 (Touch preparations fromprimary melanoma (a), lymph node metastasis (b), cutaneous metastasis(c), nevus (d), and a tumor free sentinel lymph node (e) surgicallyremoved from patients were analysed by in situ hybridisation with probesspecific for nucleotide sequences of the pol gene (POL), melanomainhibiting activity gene (MIA), and the influenza virus nucleoprotein(FLU). Nucleotide sequences of the pol and the melanoma inhibitingactivity genes are found in primary melanoma and lymph node andcutaneous metastases but not in the nevus and tumor free lymph node),high copy numbers of the pol sequence were found in tumor cells of allmelanoma preparations tested. In comparison, cells derived from thelymph node and the benign nevus of healthy individuals were negative. Aprobe specific for the nucleoprotein gene of influenza virus was alwaysnegative. As a positive control, a probe recognising themelanoma-inhibiting activity gene was used. The rate of tumor cellsexpressing the pol gene was in the range of 60-90%, a percentage similarto the one obtained with the melanoma-inhibiting activity specificprobe. The observation that the sequences are not found in all tumorcells might be due to the sensitivity of the assay and variations in theexpression levels.

Example 6 Retroviral gag, cORF, and Env Proteins are Expressed inMelanomas

To determine whether HERV-specific proteins were detectable in melanomacells, immunofluorescence analysis with antisera recognising the gag,cORF and env proteins of HERV-K was performed.

Cells grown on chamber slides, as well as touch preparations from anevus and primary melanoma on coated slides (DAKO) were fixed in 4%paraformaldehyde for 20 mins, washed in PBS and dehydrated throughgraded alcohols to absolute ethanol and air dried. In addition, fivemicrometer sections prepared from routinely processed paraffin waxblocks of a nevus and melanoma were placed on coated slides (DAKO). Thetissues were dewaxed in xylene, rehydrated by sequential immersion ingraded ethanols and PBS and subsequently permeabilized by microwavetreatment.

Immunofluorescence staining was performed by incubating chamber slides,touch preparations, and paraffin sections with HERV-K-specificantibodies at a dilution of 1:100 for 1 h at 37° C. in a humidified box,followed by washing three times with PBS and subsequent incubation withAlexafluor-488-conjugated antibodies at a dilution of 1:200 for 1 h at37° C. Counterstaining was performed by mounting in Vectashieldcontaining DAPI (Vector). Preparations were analysed by using a Zeissfluorescence microscope with appropriate filters.

FIG. 4 a shows that the melanoma cell lines Mel-Juso and SK-Mel28express the gag, cORF and env proteins. The percentage of gag-expressingcells was in the range of 1-10%, cORF was found to be present in about20%, while expression of the env protein was detected in about 10% ofthe analysed cells. Expression of gag and env was found in thecytoplasm, while cORF was mainly found in the nucleus. In contrast,cultured human melanocytes (Nhem) and Vero cells did not react with anyHERV-K-specific antisera. None of the corresponding preimmune-sera wasreactive with any of the cells tested.

Immunofluorescence analysis was performed with touch preparation of aprimary melanoma (first column); touch preparation of a nevus (secondcolumn); paraffin preparation primary melanoma (third column); paraffinpreparation nevus (fourth column). Retroviral proteins are present inthe melanoma preparations but absent from nevus tissue.

It was then analysed whether these proteins are also expressed inprimary melanoma. FIG. 4 b shows that gag, cORF, and env were detectedin touch preparations and paraffin sections of a primary melanoma butnot in the corresponding preparations of a nevus.

Example 7 Melanoma Cell Derived Particles are Infectious

Supernatants of approximately 10⁸ melanoma cells were loaded on 30%sucrose cushions and centrifuged at 28K. The resulting pellets werewashed and resuspended in 500 μl PBS. For infection studies human 293cells were grown to approximately 30% confluency and exposed overnightto the particle preparations in the presence of 8 μg/ml polybrene. Ascontrols, cells were treated with particle preparations that were heatinactivated 30 min at 60° C. or mock-treated. 293 cells were exposed toparticle preparations from the melanoma cell line SKMel28.

The human epithelial cell line 293 is a well established target forretrovirus infection. Immunofluorescence with HERV-K specific gagantibodies was performed three weeks post infection (p.i.). Controlcells were mock-treated. 293 cells were treated with themelanoma-derived viral particles, and demonstrate that upon treatmentthey express the HERV-K specific gag-protein (FIG. 5 a). Moreover,expression of the retroviral proteins correlated with the level ofpelletable RT-activity in the supernatants (FIG. 5 b), suggesting thatthe 293 cells were infected with the melanoma-derived particles.However, due to the high copy numbers of various HERV sequences presentin the human genome it was not possible to demonstrate that expressionof proteins and production of RT-containing particles was derived fromde novo integrated retroviral DNA into the hosts genome. Therefore thisquestion was addressed in bovine MDBK cells.

Example 8 Infection of MDBK Cells

It was tested whether the melanoma-derived particles are infectious. Dueto the high copy numbers of various HERV sequences present in the humangenome it is difficult to demonstrate in human cells whether virusparticles produced are derived from endogenous or exogenous virus. Thisquestion was therefore addressed in bovine MDBK cells which are free ofviruses that are homologous to HERV-K. MDBK cells were exposed tomelanoma derived particle preparations and passaged at a ratio of 1:10.Supernatants of these cells were analysed by RT-PCR with pol-specificHERV primers as described below:

Cell free supernatants of approximately 10⁸ 518A2 melanoma cells wereloaded on 20% sucrose cushions and pelleted at 28K in a Beckmann SW28rotor. The resulting pellets were washed and resuspended in 500 μl PBS.For infection studies bovine MDBK cells were grown to approximately 30%confluency and exposed overnight to 10 μl aliquots of the particlepreparations in the presence of 0.8% polybrene. As controls, cells weretreated with particle preparations that were heat inactivated. 24 hourpost infection (p.i.) the inoculum was removed, the cells were washedtwice with PBS and incubated with normal media. Starting at 7 days p.i.the cells were continuously passaged at a ratio of 1:10 and thesupernatants were analysed for the presence of viral particles bydetecting particle-associated RNA. In addition 1.5 ml of thesupernatants from infected MDBK cells were put onto new (uninfected)MDBK cells, in the presence of polybrene. 24 hours later the cells werewashed twice with PBS and incubated for 7 days, and passaged at a ratioof 1:10. The release of particles from the cells was determined bydetecting particle-associated RNA in the supernatants by RT-PCR asdescribed below:

For detecting particles released by the cells, supernatants fromapproximately 10⁷ 518A2 or infected MDBK cells were filtered, pelleted 2hours at 28K and resuspended in 500 μl Trizol. To this suspension 100 μlchloroform were added. After vortexing and centrifugation the upperphase was precipitated with isopropanol. The precipitate was pelleted bycentrifugation and washed with 70% ethanol. The pellet was resuspended,treated with DNAse in the presence of 25 mM MgCl₂ for 45 min at 37° C.,and the DNAse was inactivated for 10 min at 65° C. After precipitationwith 96% ethanol, the pellet was washed with 70% ethanol, resuspended inreverse transcription (RT) reaction buffer and RT was performed with therandom primer p(dN)6.1/10 of the reverse transcription reaction was usedas template for PCR with the oligonucleotide primers 5′108 propol 39255′CCACTGTAGAGCCTCCTAAACCC 3′ (SEQ ID No 6) and 3′108 pol 4315 5′GCTGGTATAGTAAAGGCAAATTTTTC 3′ (SEQ ID No 7) (Codon Genetic Systems,Austria) which correspond to conserved regions within the pol gene.

10 μl of each amplification product were analysed by electrophoresis ina 1.5% agarose gel and visualised by ethidium bromide staining. Analiquot of the amplification product was cloned and sequenced.Expression of HERV-specific RNA expressed in MDBK cells was detected byRT-PCR of total genomic RNA derived from approximately 10⁷ cells in thesame manner.

FIG. 6 shows the result after the first passage. Lanes 1-6: Afterinfection cells were passaged 1:10 and supernatants were analysed byRT-PCR with pol-specific primers (+). Absence of contaminating DNA wasanalysed by omitting the RT step (−); Lane 1, no template control; lane2, heat inactivated particles; lane 3, particle preparation diluted 1:100, lane 4, 1: 10 dilution, lane 5, undiluted particle preparations;lane 6, 518A2 supernatant; M, molecular weight marker; Analysis ofgenomic DNA with pol-specific primers by PCR. Lane 1, no templatecontrol; lane 2, no template control; lane 3, MDBK DNA; lane 4, 518A2DNA, lane 5, no enzyme control. Infection with undiluted particlepreparations revealed a pol-specific amplification product lane 5, +),while infection with particle preparations that were diluted 1:10 (lane4+), diluted 1:100 (lane 3+) or were heat inactivated (lane 2+) did not.None of the reactions revealed a signal when the RT step was omitted,excluding that the signals were derived from genomic DNAs contaminations(lanes 2-6, −). The presence of particle associated viral sequences inthe supernatant of the melanoma-derived particle exposed MDBK cellsindicates that melanoma-derived particles are capable to infect bovinecells.

Sequence analysis of the amplification product of lane 5 revealed thenucleotide sequence5′CCACTGTAGAGCCTCCTAAACCCATACCATTAACTTGGAAAACAGAAAAACCGGTGTGGGTAAATCAGTGGCCGCTACCAAAACAAAAACTGGAGGCTTTACATTTATTAGCAAATGAACAGTTAGAAAAGGGTCATATTGAGCCTTCGTTCTCACCTTGGAATTCTCCTGTGTTTGTAATTCAGAAGAAATCAGGCAAATGGCATATGTTAACTGACTTAAAGGCCGTAAACGCCGTAATTCAACCCAT 3′ (SEQ IDNo 8). This sequence is highly homologues (98%) to the sequence fromHERV-K 108.

Example 9 Supernatants from Infected MDBK Cells Contain InfectiousParticles

Cell free supernatants from cells exposed to undiluted melanoma particlepreparations were put onto new (uninfected) MDBK cells which weresubsequently passaged 3 times at a ratio of 1:10

FIG. 7 shows the transfer of supernatants from melanoma-particle exposedMDBK cells to uninfected MDBK cells. 24 hours later cells were washedtwice with PBS, incubated for one week, and subsequently passaged at aratio of 1:10. FIG. 7 a shows pelleted supernatants from these cellswhich were analysed for release of particle-associated RNA by RT-PCRwith pol-specific primers (lanes 1, 3, 5, 7). As negative controlssupernatants from MDBK cells exposed to heat inactivated particles weretransferred to MDBK cells which were treated and analysed in the samemanner (lanes 2, 4, 6, 8). Lanes 1 and 2: 5 days after transfer; lanes 3and 4: passage 1; lanes 5 and 6: passage 2; lanes 7 and 8: passage 3;lane M: molecular weight marker (Boehringer Mannheim VIII); lane 9:negative control: pelleted supernatant from uninfected MDBK cells; lane10: positive control: pelleted supernatants from 518A2 cells; FIG. 7 bshows the expression of HERV-specific RNAs which was analysed by RT-PCRfrom whole genomic RNA of MDBK cells from passage 1 with pol-specificprimers. Lane 1: MDBK cells treated with supernatants from infected MDBKcells; lane 2: MDBK cells treated with supernatants from MDBK cellsexposed to heat inactivated melanoma-derived particle preparations; laneM, molecular weight marker (Boehringer Mannheim VIII); lane 3, negativecontrol: PCR of genomic DNA derived from uninfected MDBK cells; lane 4,positive control: PCR of genomic DNA derived from 518A2 cells; lane 5:non-template control. Lanes 1, 3, 5, and 7 correspond to passages 0, 1,2, and 3 respectively. Lanes 2, 4, 6, and 8 are the negative controlsand correspond to passages 0, 1, 2, and 3 of cells treated withsupernatants from cells exposed to heat inactivated particlepreparations.

Shown is the presence of particle associated RNA as indicated by thepresence of a pol-specific sequences. Pol-specific signals weredetectable after passages 0, 1, and 3, suggesting the presence of viralparticles (lanes 1, 3, and 7). Analysis of passage number 2 did notreveal a pol-signal which might be due to fluctuating production levelsof viral particles (lane 5). Transfer of supernatants from MDBK cellsthat were treated with heat inactivated particle preparations did notresult in the release of particles (lanes 2, 4, 6, and 8). PCR alone didnot reveal pol-specific signals, indicating that the signals obtained byRT-PCR are derived from RNA. The result that transfer of the supernatantfrom infected MDBK cells to uninfected MDBK cells resulted in therelease of particle associated viral RNA, indicates that infection withmelanoma derived particles of MDBK cells is productive.

These data show that retroviral sequences and infectious particles areexpressed in melanoma cells.

Partial sequence analysis, immunoblotting and immuno-electron microscopystudies show the particles belong to the HERV-K family. In contrast toother known HERV-K like viruses, which lack infectivity, the envprecursor appears to be cleaved in the melanoma derived particles,resulting in approximately equimolar amounts of the precursor and theputative transmembrane domain. Cell-free particle preparations from themelanoma cells were able to infect both human and bovine cells, asindicated by the appearance of the viral proteins gag, cORF, and env,and pelletable RT-activity in the supernatants of infected cells.Moreover, the fact that human pol and gag sequences in the genome of theinfected bovine MDBK cells were detected, indicates that themelanoma-associated viruses are capable of de novo integration of theirgenome. This observation also suggests the presence of a functionalintegrase in the viral particles.

FIG. 8 a shows the pCR4-Topo vector into which the MERV gag, pro, poland env sequences isolated from the above described infected bovinecells were inserted. The primers used in the above examples are shown inthe boxes of each sequence of vectors shown in FIG. 8 b. These vectorswere deposited on 26 Sep. 2001 at the DSMZ under the names MERV-env,MERV-gag, MERV-prt and MERV-pol according to the Budapest Treaty.

1. An isolated infectious or attenuated human endogenous retroviruscomprising an RNA polynucleotide molecule capable of being reversetranscribed into a sequence that is or is complementary to a sequencehaving at least 99% identity to full length SEQ ID NO:
 1. 2. Theinfectious or attenuated human endogenous retrovirus of claim 1, furtherdefined as encoded by a polynucleotide molecule comprising a sequencehaving at least 99% identity to full length SEQ ID NO: 1.